The aim of this research is to delineate, in different intestinal epithelial cell types, the various subcellular membrane-related mechanisms which transport calcium. These membrane systems may be important in the physiological regulation of cytoplasmic free calcium as well as in transcellular calcium absorption. Membrane calcium-pumps can act by (i) extrusion of calcium from the cell across the baso-lateral membrane, and (ii) by sequestration of calcium within intracellular storage sites, which, in other tissues, occur in the endoplasmic reticulum, mitochondria and the Golgi apparatus. Evidence suggests such stores are involved in the distal small intestine in hormonal control of water and electrolyte secretion and absorption and thus are important in the pathogenesis and treatment of diarrheal diseases. In the proximal small intestine, calcium-pumps are involved in calcium transport across the epithelial cell during vitamin D-dependent active calcium absorption and so may be relevant in osteomalacia and senile osteoporosis. It is planned to investigate the differences in the subcellular membrane calcium-transport mechanisms in the proximal:distal intestinal gradient of function, and also in the crypt:villus-tip gradient of maturation. Previous work has studied vitamin D-dependent calcium binding to intestinal Golgi membranes and preliminary studies have shown the presence of ATP-dependent calcium transport in intestinal Golgi membranes. The specific aims of the proposed research are:- (1) preparation of subcellular membrane fractions, and characterization with respect to markers for Golgi, baso-lateral, and endoplasmic reticulum membranes, using cells isolated from (a) proximal or distal segments or rat small intestine, and (b) villus-tip or crypt regions of the segments; (2) determination of ATP-dependent calcimm uptake by these intestinal membrane vesicle fractions, and investigation of factors known in other tissues to be capable of inhibiting or stimulating membrane calcium transport; and (3) investigation of the effects of hormonal agents including 1,25-dihydroxycholecalciferol, and various agents affecting secretion or absorption, on ATP-dependent calcium uptake in membrane vesicles representing different subcellular components. The planned research will be a further step toward the long-term goal of understanding the regulation of intestinal cellular absorptive processes.